TssM is essential for virulence and required for type VI secretion in Ralstonia solanacearum

Type VI secretion system is a recently discovered secretion system (T6SS) in Gram-negative bacteria. Genomic analysis indicated that Ralstonia solanacearum carry T6SS gene clusters, but up to date its characterisation and function remain unclear. In this study, Rsp0763 of R. solanacearum strain GMI1000, which is an orthologue of T6S component tssM and designated as tssM(RS), was mutated by insertion of a gentamicin (Gm) resistant cartridge. Subsequent proteome analyses revealed 15 proteins specifically secreted in GMI1000 strain and 10 specifically secreted in the GMI1000 Delta tssM(RS) strain. The secretion of 3 proteins was increased in GMI1000, and 10 proteins decreased in abundance in GMI1000 Delta tssM(RS) strain. Further investigation showed that knockout of tssM(RS) resulted in defect of biofilm formation. Additionally, expression of the flagella operon is decreased in the tssM(RS) mutant, leading to decreased motility. More importantly, virulence of GMI1000 Delta tssM(RS) on tomato plants was significantly attenuated in comparison to that of the wild-type strain GMI1000.